This invention is related to the field of microbiology. Specifically, the invention relates to alteration of metabolic pathways in E. coli and other bacteria.
Bacteria grown in the laboratory have three stages of growth: the lag phase, in which little change in the number of viable cells occurs; the log phase, in which exponential increase in numbers occurs;.and the stationary phase, in which increase ceases. During the transition into stationary phase, bacteria acquire numerous new physiological properties which enhance their ability to compete and survive under suboptimal conditions. For reviews, see Kolter (1992) ASM News 58:75-79; Matin (1991) Mol. Microbiol. 5:3-10; Matin et al. (1989) Ann. Rev. Microbiol. 43:293-316; and Siegele et al. (1992) J. Bacteriol. 174:345-348. A few global regulatory factors mediate many of the extensive changes in gene expression that occur as E. coli enters the stationary phase. In E. coli, for example, the induction of several genes and operons requires a putative sigma factor known as rpoS or katF. Bohannon et al. (1991) J. Bacteriol. 173:4482-4492; Lange et al. (1991) Mol. Microbiol. 5:49-51; Matin (1991); and Schellhorn et al. (1992) J. Bacteriol. 174:4769-4776.
One of the metabolic pathways transcriptionally activated in stationary phase mediates glycogen biosynthesis. The accumulation of glycogen in the early stationary phase reflects at least two levels of control: allosteric regulation of the committed step of the biochemical pathway; and enhanced expression of the structural genes for the pathway. Expression of this pathway does not require rpoS for expression. Bohannon et al. (1991).
The essential enzymes of the glycogen pathway are glgC (encoding ADPglucose pyrophosphorylase [EC 2.7.7.27]) and glgA (encoding glycogen synthase [EC 2.4.1.21]), which are apparently cotranscribed in an operon, glgCAY. Romeo et al. (1990) Curr. Microbiol. 21:131-137; and Romeo et al. (1989) J. Bacteriol. 171:2773-2782. The operon also includes the gene glgY or glgP, which encodes the catabolic enzyme glycogen phosphorylase [E.C 2.4.1.1]. Romeo et al. (1988) Gene 70:363-376; and Yu et al. (1988) J. Biol. Chem. 263:13706-13711. Four stationary-phase-induced transcripts have been mapped within the 0.5 kb upstream noncoding region of the glgC gene from E. coli, implying a complex transcriptional regulation of glgCA.
Located upstream of glgCAY is another operon, glgBX, also encoding genes involved in the glycogen pathway. The gene glgB encodes glycogen branching enzyme [EC 2.4.1.18] and is transcribed independently of glgCA. Baecker et al. (1986) J. Biol. Chem. 261:8738-8743; Romeo et al. (1988); Preiss et al. (1989) Adv. Microb. Physiol. 30:183-233; and Romeo et al. (1989).
The gene csrA or xe2x80x9ccarbon storage regulatorxe2x80x9d is a trans-acting factor which effects potent negative regulation of glycogen biosynthesis. Romeo et al. (1993a) J. Bacteriol. 175:4744-4755; and Romeo et al. (1993b) J. Bacteriol. 175:5740-5741. CsrA is a global regulator which controls numerous genes and enzymes of carbohydrate metabolism. In E. coli K-12, it exerts pleiotropic effects, acting as a negative regulator of glycogen biosynthesis, gluconeogenesis and glycogen catabolism, as a positive factor for glycolysis, and affecting cell surface properties. Romeo et al. (1993a); Liu et al. (1995) J. Bacteriol. 177:2663-2672; Sabnis et al. (1995) J. Biol. Chem. 270:29096-29104; and Yang et al. (1996) J. Bacteriol. 178:1012-1017.
Csr is the third system discovered to be involved in the regulation of glgCA-mediated glycogen biosynthesis and the only one known to down-regulate the expression of this operon. The other two involve cyclic AMP (cAMP)/cAMP receptor protein and guanosine 3xe2x80x2-bisphosphate 5xe2x80x2-bisphosphate (ppGpp), which are positive regulators of glgCA. Romeo et al. (1990); Romeo et al. (1989); Bridger et al. (1978) Can. J. Biochem. 56:403-406; Dietzler et al. (1979) J. Biol. Chem. 254:8308-8317; Dietzler et al. (1977) Biochem. Biophys. Res. Commun. 77:1459-1467; Leckie et al. (1983) J. Biol. Chem. 258:3813-3824; Leckie et al. (1985) J. Bacteriol. 161:133-140; and Taguchi et al. (1980) J. Biochem. 88:379-387. The physiological role played by these three systems may be to establish an intrinsic metabolic capacity for glycogen synthesis in response to nutritional status. The effects of other regulatory factors, such as the allosteric effectors fructose-1,6-bisphosphate and AMP, may be superimposed upon this intrinsic metabolic capacity.
glgCA expression does not appear to be regulated by other global systems such as the nitrogen starvation system, mediated by NtrC and NtrA or "sgr"54; heat shock, mediated by "sgr"32; or the katF-dependent system. Preiss et al. (1989); Romeo et al. (1989); and Hengge-Aronis et al. (1992) Mol. Microbiol. 6:1877-1886.
A homolog of csrA in the pathogenic Erwinia species is rsmA (repressor of stationary phase metabolites). Chatterjee et al. (1995) Appl. Environ. Microbiol. 61:1959-1967; and Cui et al. (1995) J. Bacteriol. 177:5108-5115. rsmA has a role in the expression of several virulence factors of soft rot disease of higher plants, including pectinase, cellulase and protease activities. rsmA may also modulate the production of the quorum-sensing metabolite N-(3-oxohexanoyl)-L-homoserine lactone. Homologs of this metabolite are secreted by numerous Gram-negative bacteria, where they activate the expression of a variety of genes in response to cell density, as reviewed in Fuqua et al. (1994) J. Bacteriol. 176:269-275; and Swift et al. (1996) TIBS 21:214-219. Widespread phylogenetic distribution of csrA homologs among eubacteria points to a broad significance and ancient origin for this regulatory system in this group of organisms. White et al. (1.996) Gene 182:221-223; and Romeo (1996) Res. Microbiol. 147:505-512.
The csrA gene product or protein (CsrA) is a 61 amino acid protein containing a conserved RNA-binding motif and apparently mediates its regulatory activity via a cis-acting region located close to or overlapping the glgC ribosome binding site. Liu et al. (1995). CsrA strongly inhibits glycogen accumulation and affects the ability of cells to utilize certain carbon sources for growth. The down-regulated expression of csrA and CsrA can be useful for enhancing expression of products produced by alternative pathways. Such products include, but are not limited to, antibiotics, metabolites, organic acids, amino acids and a wide variety of industrially important compounds produced in bacterial fermentation systems.
As an example, down-regulating csrA expression can be used to increase the production of aromatic amino acids (e.g., tyrosine, phenylalanine, and tryptophan). These amino acids, which are commercially produced using E. coli cultures, have numerous uses, including the production of aspartame (Nutrisweet(trademark)). The TRI-5 mutation in csrA (csrA::kanR) causes overexpression of the genes pckA [encoding phosphoenolpyruvate (PEP) carboxykinase] and pps (phosphoenolpyruvate synthase), thereby raising production of PEP. PEP is in turn a precursor of aromatic amino acids and other metabolic products.
Further xe2x80x9cmetabolic engineeringxe2x80x9d can lead to even greater yields of desired amino acids or other products. In addition to being an amino acid precursor, PEP is a precursor of glucose via gluconeogenesis. Glucose is, in turn, a precursor of glycogen. Gluconeogenesis and glycogen synthesis are elevated in csrA mutants and would compete for the synthesis of aromatic amino acids. Therefore, in order to further increase carbon flow into the desired products (e.g., amino acids), engineering of gluconeogenesis, glycogen biosynthesis and possibly other pathways is desirable. A mutation in fbp, which encodes fructose-1,6-bisphosphatase, prevents gluconeogenesis from proceeding beyond the synthesis of fructose-1,6-bisphosphate. A mutation in glgC (ADP-glucose pyrophosphorylase) or glgA (glycogen synthase) further blocks residual glucose or glucose derivatives obtained from the media or generated within the cell from being used for glycogen synthesis. Each of these mutations is already known and can be introduced into a cell by methods known in the art. Further enhancement of the synthesis of a single aromatic amino acid can be achieved by introducing mutations which block the synthesis of other amino acids.
Thus, CsrA can be utilized in the context of the biochemical pathways known in the art to increase expression of desired compounds in bulk bacterial cultures.
All of the literature and patents cited herein, supra and infra, are hereby incorporated herein by reference.
The invention encompasses the E. coli csrB gene and RNA encoded thereby. csrB RNA binds to the global regulatory protein CsrA and antagonizes its ability to down-regulate expression of metabolic products.
This invention also encompasses methods using the csrB gene and csrB RNA to modulate activity of CsrA and, in turn, to regulate expression of metabolic products in bacterial cultures.
This invention also encompasses compositions comprising csrB polynucleotides and fragments and derivatives thereof, in combination with CsrA polypeptides and fragments and derivatives thereof.
This invention also encompasses antibodies to csrB polynucleotides or complexes formed between csrB polynucleotides and CsrA polypeptides.
Accordingly, in one aspect, the invention provides an isolated csrB polynucleotide.
In one embodiment the invention provides the sequence of the polynucleotide with at least 70% identity to that depicted in FIG. 1 (SEQ ID NO:1), and which comprises at least one binding site for CsrA protein or a complement of a binding site for CsrA protein.
In another embodiment, the invention provides the polynucleotide depicted in FIG. 1 (SEQ ID NO: 1).
In another embodiment, the invention provides a composition comprising a csrB polynucleotide and CsrA wherein the ratio of csrB polynucleotide to CsrA is about 1:18.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell by altering genetic expression or CsrA-binding activity of csrB, wherein CsrA-binding activity of csrB is altered by mutating csrB.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB, wherein a result of altered genetic expression of csrB is a change in the level of production of a metabolic compound, wherein the level of production is at least partially regulated by CsrA.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB, wherein a result of altered genetic expression of csrB is a change in glycogen biosynthesis or gluconeogenesis.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB, wherein a result of altered genetic expression of csrB is a change in the level of production of a metabolic compound, wherein the level of production is at least partially regulated by CsrA, wherein the metabolic compound is an amino acid.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB, wherein expression of the csrB gene is increased.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB wherein expression of the csrB gene is decreased.
In another embodiment, the invention provides a method of altering the metabolism or structural or functional properties of a cell comprising altering genetic expression or CsrA-binding activity of csrB wherein expression of the csrB gene is under inducible control.
In another embodiment, the invention provides an antibody capable of binding to a composition comprising a CsrA and a csrB polynucleotide.
In another embodiment, the invention provides a method of modulating the level of production of a metabolic compound, wherein the level of production is at least partially regulated by CsrA, comprising the step of binding to CsrA an antibody capable of binding to a composition comprising CsrA and a csrB polynucleotide.
In another embodiment, the invention provides a vector comprising a csrB gene, further comprising a csrA gene
In another embodiment, the invention provides a vector comprising a csrB gene, further comprising a csrA gene, wherein the csrB gene and csrA gene are controlled by different inducible promoters.
In another embodiment, the invention provides a method of modulating the level of production of a metabolic compound, wherein the level of production is at least partially controlled by CsrA, comprising introducing a vector comprising a csrB gene, further comprising a csrA gene, into a host cell; and raising the level of production of the metabolic compound by inducing expression of csrB.
In another embodiment, the invention provides a method of modulating the level of production of a metabolic compound, wherein the level of production is at least partially controlled by CsrA, comprising introducing a vector comprising a csrB gene, further comprising a csrA gene, into a host cell; raising the level of production of the metabolic compound by inducing expression of csrB; and decreasing the level of production of the metabolic compound by inducing expression of csrA.